1. Field of the Invention:
The present invention relates to a biosensor that can easily quantify a substrate (a specific component) in a sample liquid with speed and accuracy, and to a method and a device for quantifying a substrate by using the biosensor. More particularly, the invention relates to a biosensor that can quantify a substrate in a sample liquid by electrochemically measuring the amount of an electron acceptor that has been reduced with an electron generated in a reaction between the substrate in the sample liquid and an oxidoreductase that specifically reacts with the substrate, and to a method and a device for quantifying a substrate by using the biosensor.
2. Description of the Related Art:
The optical rotation method, the colorimetric method, the reductimetry method and other methods using different kinds of chromatographies have been developed as methods for quantitative analysis of saccharides such as sucrose and glucose. However, none of these methods can provide high accuracy due to the relatively low specificity against saccharides. Among these methods, the optical rotation method is easy to operate but is largely influenced by the operating temperature. Therefore, it is not appropriate for common use at home and the like.
Various types of biosensors utilizing a specific catalysis of an enzyme have been recently developed. As an example of methods for quantifying a substrate in a sample liquid, a method for quantifying glucose will now be described. For electrochemically quantifying glucose, a method using glucose oxidase (EC1.1.3.4; hereinafter referred to as GOD) and an oxygen electrode or a hydrogen peroxide electrode is generally known (for example, "Biosensor" edited by Shuichi Suzuki, Kodansha Kabushiki Kaisha).
GOD selectively oxidizes .beta.-D-glucose into D-glucono-.delta.-lactone by using oxygen as an electron acceptor. In an oxidation reaction using GOD in the presence of oxygen, oxygen is reduced to hydrogen peroxide. In the aforementioned method, the amount of reduced oxygen is measured by using an oxygen electrode, or the amount of increased hydrogen peroxide is measured by using a hydrogen peroxide electrode. Since the amounts of the reduced oxygen and the increased hydrogen peroxide are in proportion to the content of glucose in the sample liquid, glucose can be quantified based on the amount of the reduced oxygen or the increased hydrogen peroxide.
The above-mentioned method, however, has a problem in that the measurement result is largely affected by the concentration of oxygen in the sample liquid, as can be presumed from the reaction process. Furthermore, when no oxygen is contained in the sample liquid, the above-mentioned method cannot be adopted.
In order to solve this problem, a new type of glucose sensor has been developed, in which an organic compound such as potassium ferricyanide, a ferrocene derivative and a quinone derivative or a metal complex is used as an electron acceptor instead of oxygen. In this type of sensor, the reduced form of an electron acceptor resulting from the enzymatic reaction is oxidized on an electrode, and the concentration of glucose contained in a sample liquid is obtained based on the amount of the measured oxidation current. By using an organic compound or a metal complex as an electron acceptor instead of oxygen, it is possible to hold a known amount of GOD and its electron acceptor on an electrode stably and accurately, so as to form a reaction layer. In this case, the reaction layer can be integrated with the electrode in a substantially dry state. Therefore, a disposable glucose sensor based on this technique has recently come to public notice. In this disposable glucose sensor, the concentration of glucose in a sample liquid can be easily measured with a measuring device merely by introducing the sample liquid into the sensor removably connected to the measuring device. Such a method can be applied not only to the quantification of glucose but also to the quantification of any other substrate contained in a sample liquid.
When a substrate in a sample liquid is quantified by such a method, a two-electrode system sensor having a working electrode and a counter electrode is generally used.
In the measurement using such a two-electrode system sensor, before applying a voltage between the working electrode and the counter electrode to obtain a current response, a voltage is generally applied between the working electrode and the counter electrode so as to detect a liquid junction based on the change of a resistance value between these electrodes. In such a case, the interface between the electrodes can be varied by the application of the voltage, resulting in affecting the measurement. Furthermore, the resistance value between the working electrode and the counter electrode can be sometimes varied to start the measurement before supplying a sufficient amount of a sample liquid to the electrode system. This also affects the measurement.